Novel thyroid peroxidase autoantibody immunoassay

ABSTRACT

Disclosed herein are immunoassays for detecting an anti-thyroid peroxidase antibody in a biological sample from a subject and/or diagnosing a thyroid disease in a subject. The disclosed immunoassays employ a recombinant cynomolgus monkey thyroid peroxidase (rTPO) and assess the level of anti-thyroid peroxidase antibody-induced formation or disruption of complexes comprising a solid support and the rTPO.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority of U.S. Provisional Application No.62/693,434 filed Jul. 2, 2018, which is incorporated by reference hereinin its entirety.

TECHNICAL FIELD

Disclosed herein are methods of detecting an anti-thyroid peroxidaseantibody in a biological sample from a subject and methods of diagnosinga thyroid disease in the subject.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 12, 2021, isnamed 2018P03766WO_ST25.tx and is 7,533 bytes in size.

BACKGROUND

Thyroid peroxidase (TPO) is a membrane bound glycoprotein found in theapical membrane of thyroid follicular cells. TPO oxidizes iodide ionsfor addition onto thyroglobulin tyrosine residues for production of keythyroid hormones T₃ and T₄. TPO is also an autoantigen in thyroiddiseases, including, for example, Hashimoto's thyroiditis, Graves'disease, atrophic thyroiditis, primary mixedema, and postpartumthyroiditis in women. Elevated levels of anti-TPO autoantibody are arisk factor for thyroid diseases.

Current immunoassays for detecting patient anti-TPO autoantibody utilizehuman-derived TPO, which is obtained from human cadaver thyroid tissue.Human-derived TPO, however, is costly, difficult to purify, andlow-yield, and can vary significantly from lot to lot. The currentimmunoassays further require multiple non-human secondary antibodies.

SUMMARY

Disclosed herein are methods of detecting an anti-thyroid peroxidaseantibody in a biological sample from a subject. The methods can compriseincubating the biological sample from the subject with a solid support,an unlabeled recombinant cynomolgus monkey thyroid peroxidase (rTPO),and a labeled cynomolgus monkey rTPO. In the presence of theanti-thyroid peroxidase antibody, a complex comprising the labeledcynomolgus monkey rTPO, the unlabeled cynomolgus monkey rTPO, and thesolid support is formed. The methods further comprise detecting thecomplex, the presence of which indicates the presence of theanti-thyroid peroxidase antibody in the biological sample. In someembodiments, the methods can be an “antigen bridge” assay as describedherein.

Also provided are methods of detecting an anti-thyroid peroxidaseantibody in a biological sample from a subject, the methods comprisingincubating the biological sample from the subject with a solid support,a cynomolgus monkey rTPO, and an anti-human secondary antibody. In thepresence of the anti-thyroid peroxidase antibody, a complex comprisingthe solid support, the cynomolgus monkey rTPO, and the anti-humansecondary antibody is formed. The methods further comprise detecting thecomplex, the presence of which indicates the presence of theanti-thyroid peroxidase antibody in the biological sample. In someembodiments, the methods can be an “rTPO capture” assay as describedherein. In some embodiments, the methods can be an “IgG class capture”assay as described herein.

Further provided are methods of detecting an anti-thyroid peroxidaseantibody in a biological sample from a subject, the methods comprisingincubating the biological sample from the subject with a solid support,an unlabeled anti-TPO antibody, a cynomolgus monkey rTPO, and a labeledanti-TPO antibody and detecting the anti-thyroid peroxidase antibody,the detecting comprising analyzing a decrease in the formation of acomplex comprising the solid support, the unlabeled anti-TPO antibody,the cynomolgus monkey rTPO, and the labeled anti-TPO antibody. Thepresence of an anti-thyroid peroxidase antibody in the biological sampledecreases formation of a complex comprising the solid support, theunlabeled anti-TPO antibody, the cynomolgus monkey rTPO, and the labeledanti-TPO antibody. The presence of complex is inversely proportional tothe presence of the anti-thyroid peroxidase antibody in the biologicalsample. In some embodiments, the methods can be a “competition” assay oran “inhibition” assay, as described herein.

Further disclosed herein are methods of diagnosing a thyroid disease ina subject. The methods can comprise incubating a biological sample fromthe subject with a solid support, an unlabeled recombinant cynomolgusmonkey thyroid peroxidase (rTPO), and a labeled cynomolgus monkey rTPO.In the presence of an anti-thyroid peroxidase antibody in the biologicalsample, a complex comprising the labeled cynomolgus monkey rTPO, theunlabeled cynomolgus monkey rTPO, and the solid support is formed. Themethod further comprises diagnosing the subject with the thyroid diseaseif the complex is formed.

Methods of diagnosing a thyroid disease in a subject comprisingincubating a biological sample from the subject with a solid support, acynomolgus monkey rTPO, and an anti-human secondary antibody are alsoprovided. In the presence of an anti-thyroid peroxidase antibody in thebiological sample, a complex comprising the solid support, thecynomolgus monkey rTPO, and the anti-human secondary antibody is formed.The method further comprises diagnosing the subject with the thyroiddisease if the complex is formed.

Further provided are methods of diagnosing a thyroid disease in asubject comprising incubating a biological sample from a subject with asolid support, an unlabeled anti-TPO antibody, a cynomolgus monkey rTPO,and a labeled anti-TPO antibody, and diagnosing the subject with thethyroid disease if the formation of a complex comprising the solidsupport, the unlabeled anti-TPO antibody, the cynomolgus monkey rTPO,and the labeled anti-TPO antibody is decreased. The presence of ananti-thyroid peroxidase antibody in the biological sample decreasesformation of a complex comprising the solid support, the unlabeledanti-TPO antibody, the cynomolgus monkey rTPO, and the labeled anti-TPOantibody.

Recombinantly produced thyroid peroxidase (rTPO) comprising the aminoacid sequence of SEQ ID NO: 1 is also disclosed, as are cDNA moleculesencoding the recombinantly produced rTPO.

Further disclosed herein are kits. In some embodiments, the kitscomprise a solid support, an unlabeled cynomolgus monkey rTPO, and alabeled cynomolgus monkey rTPO.

Alternatively, the kits can comprise a solid support, a cynomolgusmonkey rTPO, and an anti-human secondary antibody.

In some embodiments, the kits comprise a solid support, a cynomolgusmonkey rTPO and an anti-TPO antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is furtherunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the disclosed methods and kits, there are shownin the drawings exemplary embodiments of the methods and kits; however,the methods and kits are not limited to the specific embodimentsdisclosed. In the drawings:

FIG. 1 illustrates an exemplary reaction scheme for one embodiment ofthe disclosed methods, referred to herein as the antigen bridgeimmunoassay.

FIG. 2 illustrates an exemplary reaction scheme for one embodiment ofthe disclosed methods, referred to herein as the IgG class captureimmunoassay.

FIG. 3 illustrates an exemplary reaction scheme for one embodiment ofthe disclosed methods, referred to herein as the rTPO captureimmunoassay.

FIG. 4 illustrates an exemplary reaction scheme for one embodiment ofthe disclosed methods, referred to herein as the competition/inhibitionimmunoassay.

FIG. 5 illustrates the results from an exemplary rTPO lot-to-lot purityand immunoreactivity assessment. Panel A shows a gel stained with GelCode Blue (ThermoFisher 25590); panel B shows a nitrocellulose membraneprobed with a mouse anti-TPO monoclonal antibody followed by a goatanti-mouse IgG-HRP secondary. 4-chloro-1-naphthol (Sigma) was used todevelop the blot.

FIG. 6 illustrates the results from an exemplary rTPO isoelectric pointassessment for 4 rTPO antigen lots.

FIG. 7A and FIG. 7B illustrate the results from an exemplary assessmentof immunoassay susceptibility to deglycosylated rTPO. FIG. 7A shows arTPO antigen bridge assay and FIG. 7B shows a human IgG class captureassay built using various deglycosylated components. “Hook Ratio” refersto relative light units (RLU) of a 20,000 IU/mL sample divided by RLU ofthe highest standard at 625 IU/mL. “BKGD” refers to background RLU of anaTPO-negative serum patient pool, i.e., the lowest standard. “S08/S01”refers to RLU of the highest standard at 625 IU/mL divided by the RLU ofthe lowest standard. “SP” means “solid phase” and “LR” means “Litereagent”; “de” refers to “deglycosylation.”

FIG. 8 illustrates the results from an exemplary characterization ofrTPO complexed with autoantibodies from an anti-TPO antibody-positivedisease patient sample, with relative light units (RLU) plotted on achromatogram from size exclusion chromatography (SEC) fractions.

FIG. 9 is a chromatogram from an exemplary SEC analysis of pooledfractions from the fractions depicted in the rectangle in FIG. 8.

FIG. 10A, FIG. 10B, FIG. 10C, and FIG. 10D illustrate exemplaryMALDI-TOF spectra of isolated fractions S1, S2, S3 and S4 from FIG. 9.

FIG. 11 illustrates the results from an analysis of immunoassayperformance using native TPO and recombinant TPO (“rTPO”).

FIG. 12A, FIG. 12B, FIG. 12C, and FIG. 12D illustrate the results fromexemplary experiments performed using the disclosed immunoassays: FIG.12A) antigen bridge, FIG. 12B) IgG class capture, FIG. 12C) rTPOcapture, and FIG. 12D) competition/inhibition formats. Each assay wasperformed using the ADVIA CENTAUR® system. RLU output associated withstandardization material value was assigned from WHO 66/387 HumanAnti-thyroid Microsome Serum.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosed methods and kits may be understood more readily byreference to the following detailed description taken in connection withthe accompanying figures, which form a part of this disclosure. It is tobe understood that the disclosed methods and kits are not limited to thespecific methods and kits described and/or shown herein, and that theterminology used herein is for the purpose of describing particularembodiments by way of example only and is not intended to be limiting ofthe claimed methods and kits.

Unless specifically stated otherwise, any description as to a possiblemechanism or mode of action or reason for improvement is meant to beillustrative only, and the disclosed methods and kits are not to beconstrained by the correctness or incorrectness of any such suggestedmechanism or mode of action or reason for improvement.

Throughout this text, the descriptions refer to methods of detecting anantibody and methods of diagnosing a thyroid disease. Where thedisclosure describes or claims a feature or embodiment associated with amethod of detecting an antibody, such a feature or embodiment is equallyapplicable to the methods of diagnosing a thyroid disease. Likewise,where the disclosure describes or claims a feature or embodimentassociated with a method of diagnosing a thyroid disease, such a featureor embodiment is equally applicable to the methods of detecting anantibody.

Where a range of numerical values is recited or established herein, therange includes the endpoints thereof and all the individual integers andfractions within the range, and also includes each of the narrowerranges therein formed by all the various possible combinations of thoseendpoints and internal integers and fractions to form subgroups of thelarger group of values within the stated range to the same extent as ifeach of those narrower ranges was explicitly recited. Where a range ofnumerical values is stated herein as being greater than a stated value,the range is nevertheless finite and is bounded on its upper end by avalue that is operable within the context of the invention as describedherein. Where a range of numerical values is stated herein as being lessthan a stated value, the range is nevertheless bounded on its lower endby a non-zero value. It is not intended that the scope of the inventionbe limited to the specific values recited when defining a range. Allranges are inclusive and combinable.

When values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms anotherembodiment. Reference to a particular numerical value includes at leastthat particular value, unless the context clearly dictates otherwise.

It is to be appreciated that certain features of the disclosed methodsand kits which are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features of the disclosed methods andkits that are, for brevity, described in the context of a singleembodiment, may also be provided separately or in any subcombination.

As used herein, the singular forms “a,” “an,” and “the” include theplural.

Various terms relating to aspects of the description are used throughoutthe specification and claims. Such terms are to be given their ordinarymeaning in the art unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinitions provided herein.

The term “comprising” is intended to include examples encompassed by theterms “consisting essentially of” and “consisting of”; similarly, theterm “consisting essentially of” is intended to include examplesencompassed by the term “consisting of.”

Disclosed herein are immunoassays and methods for detecting ananti-thyroid peroxidase (anti-TPO) antibody in a biological sample froma subject and/or diagnosing a thyroid disease in a subject.

The methods of detecting an anti-thyroid peroxidase antibody in abiological sample from a subject can comprise:

-   -   a) incubating the biological sample from the subject with:        -   a solid support,        -   an unlabeled recombinant cynomolgus monkey thyroid            peroxidase (rTPO), and a labeled cynomolgus monkey rTPO,            -   wherein, in the presence of the anti-thyroid peroxidase                antibody, a complex comprising the labeled cynomolgus                monkey rTPO, the unlabeled cynomolgus monkey rTPO, and                the solid support is formed; and    -   b) detecting the complex, the presence of which indicates the        presence of the anti-thyroid peroxidase antibody in the        biological sample.

In some embodiments, the methods can comprise an “antigen bridge”immunoassay, an exemplary reaction scheme for which is illustrated inFIG. 1. A biological sample known to have, or suspected of having, ananti-TPO antibody 10, is incubated with a labeled rTPO 20 and solidsupport having an unlabeled rTPO bound thereto 30. In the absence of theanti-TPO antibody 10, the labeled rTPO 20 will not bind to or otherwiseinteract with the solid support. Thus, in the absence of the anti-TPOantibody, the labeled rTPO remains in the solution and isolation of thesolid support would not result in isolation of the labeled rTPO. Whenthe anti-TPO antibody 10 is present in the biological sample, theanti-TPO antibody 10 simultaneously binds to the unlabeled rTPO bound tothe solid support 30 and the labeled rTPO 20, thereby linking thelabeled rTPO 20 and the solid support 30 and resulting in the formationof a solid support/labeled rTPO complex 40. It is to be understood thatthe order in which the incubation takes place can be different from thatexemplified in FIG. 1. For example, the biological sample known to have,or suspected of having, an anti-TPO antibody 10 can first be incubatedwith a solid support having an unlabeled rTPO bound thereto 30 followedby incubation with a labeled rTPO 20. Alternatively, the biologicalsample known to have, or suspected of having, an anti-TPO antibody 10can simultaneously be incubated with a solid support having an unlabeledrTPO bound thereto 30 and a labeled rTPO 20.

The biological sample known to have, or suspected of having, an anti-TPOantibody 10 can be incubated in a reaction mixture for a period of timesufficient to achieve a partial reaction without allowing the reactionto achieve equilibrium, such as for about 1 minute, about 2 minutes,about 3 minutes, about 4 minutes, about 5 minutes, or less than about 10minutes. The labeled rTPO 20 can be added and incubated with thebiological sample for about 1 minute, about 2 minutes, about 3 minutes,about 4 minutes, or less than about 5 minutes. The solid support havingunlabeled rTPO bound thereto 30 can be added to the mixture ofbiological sample and labeled rTPO 20 and incubated for a period of timesufficient to achieve a partial reaction without allowing the reactionto achieve equilibrium, such as for about 1 minute, about 2 minutes,about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes,about 7 minutes, or about less than 10 minutes. In some embodiments, theincubating steps are performed in a total of about 10 minutes to about20 minutes. The subsequent detecting can be performed in less than about5 minutes. It is to be understood that the amount of time needed for theassay may vary based upon several factors including the level of theanti-TPO antibody(ies) in the biological sample and the affinity of theanti-TPO antibody(ies) for the rTPO. In some embodiments, incubating thebiological sample with the reaction mixture can be performed for aperiod of time sufficient to enable the reaction to achieve equilibrium,such as on the order of 1 or more hours. Thus, the disclosed methods canbe performed for any suitable amount of time.

The unlabeled rTPO can be directly or indirectly linked to the solidsupport. Suitable techniques for directly linking the unlabeled rTPO tothe solid support include, for example, covalent attachment, adsorption,noncovalent interaction, or combinations thereof. In some embodiments,the unlabeled rTPO can be directly linked to the solid support byN-hydroxysuccinimide (NETS) chemistry or by1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) NETS chemistry.Suitable techniques for indirectly linking the rTPO to the solid supportinclude, for example, linking through a peptide, a protein, an antibody,a linker, or a combination thereof. In some embodiments, the unlabeledrTPO can be indirectly linked to the solid support through streptavidinand biotin. For example, the unlabeled rTPO can be biotinylated and thesolid support can comprise streptavidin.

Exemplary solid supports include, but are not limited to, a columnmatrix material, a culture plate, a tube, a dish, a flask, a microtiterplate, a bead/particle, heat-killed formalin- (or otherchemically)-fixed prokaryotic or eukaryotic cells, microscope slides,ACLAR® Film, or any other optically transparent polymer, or acombination thereof. The solid support can be fully or partiallycomposed of plastic, cellulose, cellulose derivatives, nitrocellulose,glass, fiberglass, latex, or a combination thereof. In some embodiments,the solid support comprises a magnetic bead/particle. In someembodiments, the magnetic bead/particle is a paramagnetic particle(PMP). In some embodiments, the magnetic bead/particle is a latexmagnetic particle (LMP).

The label can be any suitable label known to those skilled in the art tobe useful for creating a detectable signal. Suitable detectable labelsinclude, but are not limited to, enzyme conjugates (e.g., horseradishperoxidase (HRP), alkaline phosphatase, glucose oxidase, andβ-galactosidase), fluorescent probes, radioactive isotopes,chemiluminescent compounds, bioluminescent compounds, or combinationthereof. In some embodiments, the label is an acridinium ester (“AE”) oran analog thereof. Suitable AE analogs include: dimethyl acridiniumester (DMAE), N-sulfopropyl dimethyl acridinium ester (NSP-DMAE), highquantum yield acridinium ester (HQYAE, acridinium,9-[[4-[[[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]carbonyl]-2,6-dimethylphenoxy]carbonyl]-2,7-bis(3,6,9,12,15,18-hexaoxanonadec-1-yloxy)-10-(3-sulfopropyl)-,inner salt), Zwitterionic acridinium ester (ZAE, Acridinium,9-[[4-[[[3-[[3-[[5-[(2,5-dioxo-1-pyrrolidinyl)oxy]-1,5-dioxopentyl]amino]propyl]methyl(3-sulfopropyl)ammonio]propyl]amino]carbonyl]-2,6-dimethylphenoxy]carbonyl]-10-(3-sulfopropyl)-,bis(inner salt)), N-sulfopropyl-2-isopropoxy dimethyl acridinium ester(Iso-Di-ZAE), trisulfopropyl acridinium ester (TSP-AE), or N-sulfopropyldimethyl acridinium ester with hexa(ethylene)glycol linker (HEG-GLU-AE).In some embodiments, the labeled cynomolgus monkey rTPO comprisesrTPO-NSP-DMAE. The rTPO-NSP-DMAE can be present at about 50 ng/ml toabout 2 μg/ml. In some embodiments, the rTPO-NSP-DMAE can be present atabout 220 ng/ml.

The solid support having unlabeled rTPO bound thereto and/or the labeledrTPO can be present in a buffer comprising, for example, phosphatebuffer, NaCl, EDTA, pluronic F-127, sodium azide, sorbitol, sulfhydrylmodified bovine serum, or any combinations, variations, or equivalentsthereof. In one embodiment, the buffer comprises about 100 mM phosphatebuffer, about 400 mM NaCl, about 1.9 g/L EDTA, about 0.2% (v/v) pluronicF-127, about 0.9 g/L sodium azide, about 10% sorbitol, and about 10 g/Lsulfhydryl modified bovine serum albumin.

Also provided are methods of detecting an anti-thyroid peroxidaseantibody in a biological sample from a subject comprising:

-   -   a) incubating the biological sample from the subject with:        -   a solid support,        -   a cynomolgus monkey rTPO, and        -   an anti-human secondary antibody,            -   wherein, in the presence of the anti-thyroid peroxidase                antibody, a complex comprising the solid support, the                cynomolgus monkey rTPO, and the anti-human secondary                antibody is formed; and    -   b) detecting the complex, the presence of which indicates the        presence of the anti-thyroid peroxidase antibody in the        biological sample.

The anti-human secondary antibody can be directly or indirectly linkedto the solid support and the cynomolgus monkey rTPO can comprise alabel. In some embodiments, the anti-human secondary antibody can bedirectly linked to the solid support by glutaraldehyde fixation.

In some embodiments, the methods can comprise an “IgG class capture”immunoassay, an exemplary reaction scheme for which is illustrated inFIG. 2. A biological sample known to have, or suspected of having, ananti-TPO antibody 10, is incubated with a labeled rTPO 20 and a solidsupport having an unlabeled anti-human secondary antibody bound thereto50. In the absence of the anti-TPO antibody 10, the labeled rTPO 20 willnot bind to or otherwise interact with the solid support. Thus, in theabsence of the anti-TPO antibody, the labeled rTPO remains in thesolution and isolation of the solid support would not result inisolation of the labeled rTPO. When the anti-TPO antibody 10 is presentin the biological sample, the anti-TPO antibody 10 simultaneously bindsto the unlabeled anti-human secondary antibody bound to the solidsupport 50 and the labeled rTPO 20, thereby linking the labeled rTPO 20and the solid support 50 and resulting in the formation of a solidsupport/labeled rTPO complex 60. It is to be understood that the orderin which the incubation takes place can be different from thatexemplified in FIG. 2. For example, the biological sample known to have,or suspected of having, an anti-TPO antibody 10 can first be incubatedwith a solid support having an unlabeled anti-human secondary antibodybound thereto 50 followed by incubation with a labeled rTPO 20.Alternatively, the biological sample known to have, or suspected ofhaving, an anti-TPO antibody 10 can simultaneously be incubated with asolid support having an unlabeled anti-human secondary antibody boundthereto 50 and a labeled rTPO 20.

The biological sample known to have, or suspected of having, an anti-TPOantibody 10 can be incubated in a reaction mixture for about 1 minute,about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, orless than about 10 minutes. The labeled rTPO 20 can be added andincubated with the biological sample for about 1 minute, about 2minutes, about 3 minutes, about 4 minutes, or less than about 5 minutes.The solid support having an unlabeled anti-human secondary antibodybound thereto 50 can be added to the mixture of biological sample andlabeled rTPO 20 and incubated for about 1 minute, about 2 minutes, about3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7minutes, or about less than 10 minutes. In some embodiments, theincubating steps are performed in a total of about 10 minutes to about20 minutes. The subsequent detecting can be performed in less than about5 minutes. It is to be understood that the amount of time needed for theassay may vary based upon several factors including the level of theanti-TPO antibody(ies) in the biological sample and the affinity of theanti-TPO antibody(ies) for the rTPO. Thus, the disclosed methods can beperformed for any suitable amount of time.

The cynomolgus monkey rTPO can be directly or indirectly linked to thesolid support and the anti-human secondary antibody can comprise alabel. In some embodiments, the rTPO can be directly linked to the solidsupport by N-hydroxysuccinimide (NHS) chemistry or by1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) NHS chemistry.

In some embodiments, the methods can comprise an “rTPO capture”immunoassay, an exemplary reaction scheme for which is illustrated inFIG. 3. A biological sample known to have, or suspected of having, ananti-TPO antibody 10, is incubated with a labeled anti-human secondaryantibody 70 and a solid support having an unlabeled rTPO bound thereto30. In the absence of the anti-TPO antibody 10, the labeled anti-humansecondary antibody 70 will not bind to or otherwise interact with thesolid support. Thus, in the absence of the anti-TPO antibody, thelabeled anti-human secondary antibody remains in the solution andisolation of the solid support would not result in isolation of thelabeled anti-human secondary antibody. When the anti-TPO antibody 10 ispresent in the biological sample, the anti-TPO antibody 10simultaneously binds to the unlabeled rTPO bound to the solid support 30and the labeled anti-human secondary antibody 70, thereby linking thelabeled anti-human secondary antibody 70 and the solid support 30 andresulting in the formation of a solid support/labeled anti-humansecondary antibody complex 80. It is to be understood that the order inwhich the incubation takes place can be different from that exemplifiedin FIG. 3. For example, the biological sample known to have, orsuspected of having, an anti-TPO antibody 10 can first be incubated witha solid support having an unlabeled rTPO bound thereto 30 followed byincubation with a labeled anti-human secondary antibody 70.Alternatively, the biological sample known to have, or suspected ofhaving, an anti-TPO antibody 10 can simultaneously be incubated with asolid support having an unlabeled rTPO bound thereto 30 and a labeledanti-human secondary antibody 70.

The biological sample known to have, or suspected of having, an anti-TPOantibody 10 can be incubated in a reaction mixture for about 1 minute,about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, orless than about 10 minutes. The labeled anti-human secondary antibody 70can be added and incubated with the biological sample for about 1minute, about 2 minutes, about 3 minutes, about 4 minutes, or less thanabout 5 minutes. The solid support having an unlabeled rTPO boundthereto 30 can be added to the mixture of biological sample and labeledanti-human secondary antibody 70 and incubated for about 1 minute, about2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6minutes, about 7 minutes, or about less than 10 minutes. In someembodiments, the incubating steps are performed in a total of about 10minutes to about 20 minutes. The subsequent detecting can be performedin less than about 5 minutes. It is to be understood that the amount oftime needed for the assay may vary based upon several factors includingthe level of the anti-TPO antibody(ies) in the biological sample and theaffinity of the anti-TPO antibody(ies) for the rTPO. Thus, the disclosedmethods can be performed for any suitable amount of time.

The anti-human secondary antibody or the cynomolgus monkey rTPO can bedirectly or indirectly linked to the solid support by any suitable meansknown to those skilled in the art. Suitable techniques for directlinking include, for example, covalent attachment, adsorption,noncovalent interaction, or combinations thereof. In some embodiments,the anti-human secondary can be directly linked to the solid support byglutaraldehyde fixation. Suitable means for indirect linking include,for example, linking through a peptide, a protein, an antibody, alinker, or a combination thereof. In some embodiments, the anti-humansecondary antibody or the cynomolgus monkey rTPO can be indirectlylinked to the solid support through streptavidin and biotin. Forexample, the unlabeled anti-human secondary antibody or the unlabeledrTPO can be biotinylated and the solid support can comprisestreptavidin.

Exemplary solid supports include, but are not limited to, a columnmatrix material, a culture plate, a tube, a dish, a flask, a microtiterplate, a bead/particle, heat-killed formalin- (or otherchemically)-fixed prokaryotic or eukaryotic cells, microscope slides,ACLAR® Film, or any other optically transparent polymer, or acombination thereof. The solid support can be fully or partiallycomposed of plastic, cellulose, cellulose derivatives, nitrocellulose,glass, fiberglass, latex, or a combination thereof. In some embodiments,the solid support comprises a magnetic bead/particle. In someembodiments, the magnetic bead/particle is a paramagnetic particle(PMP). In some embodiments, the magnetic bead/particle is a latexmagnetic particle (LMP).

The anti-human secondary antibody can be an IgA, IgD, IgG, IgE, or IgMisotype or a single domain format, such as a single-domain antibody fromcamelid. In some embodiments, the anti-human secondary antibody is ananti-human IgG. In some embodiments, the anti-human secondary antibodyis a commercially available anti-human secondary antibody. Aptamers thatare specific for the anti-TPO antibody can also be used.

The label can be any suitable label known to those skilled in the art tobe useful for creating a detectable signal. Suitable detectable labelsinclude, but are not limited to, enzyme conjugates (e.g., horseradishperoxidase (HRP), alkaline phosphatase, glucose oxidase, and(3-galactosidase), fluorescent probes, radioactive isotopes,chemiluminescent compounds, bioluminescent compounds, or a combinationthereof. In some embodiments, the label can be an AE or an analogthereof. Suitable AE analogs include: dimethyl acridinium ester (DMAE),N-sulfopropyl dimethyl acridinium ester (NSP-DMAE), high quantum yieldacridinium ester (HQYAE), Zwitterionic acridinium ester (ZAE),N-sulfopropyl-2-isopropoxy dimethyl acridinium ester (Iso-Di-ZAE),trisulfopropyl acridinium ester (TSP-AE), or N-sulfopropyl dimethylacridinium ester with hexa(ethylene)glycol linker (HEG-GLU-AE). In someembodiments, the labeled cynomolgus monkey rTPO comprises rTPO-NSP-DMAE.The rTPO-NSP-DMAE can be present at about 50 ng/ml to about 2 μg/ml. Insome embodiments, the rTPO-NSP-DMAE can be present at about 220 ng/ml.

The solid support having unlabeled anti-human secondary antibody orunlabeled rTPO bound thereto and/or the labeled rTPO or labeledanti-human secondary antibody can be present in a buffer comprising, forexample, phosphate buffer, NaCl, EDTA, pluronic F-127, sodium azide,sorbitol, sulfhydryl modified bovine serum, or any combinations,variations, or equivalents thereof. In one embodiment, the buffercomprises about 100 mM phosphate buffer, about 400 mM NaCl, about 1.9g/L EDTA, about 0.2% (v/v) pluronic F-127, about 0.9 g/L sodium azide,about 10% sorbitol, and about 10 g/L sulfhydryl modified bovine serumalbumin.

Also provided are methods of detecting an anti-thyroid peroxidaseantibody in a biological sample from a subject comprising:

-   -   a) incubating the biological sample from the subject with a        solid support, an unlabeled anti-TPO antibody, a cynomolgus        monkey rTPO, and a labeled anti-TPO antibody; and    -   b) detecting the anti-thyroid peroxidase antibody in the        biological sample, the detecting comprising analyzing a decrease        in the formation of a complex comprising the solid support, the        unlabeled anti-TPO antibody, the cynomolgus monkey rTPO, and the        labeled anti-TPO antibody.

Analyzing a decrease in the formation of the complex can be performed,for example, by comparing a read-out of a signal from the labeledanti-TPO antibody in a reaction mixture with the biological sample to aread-out of a signal from the labeled anti-TPO antibody in a reactionmixture without the biological sample. If the read-out of the signalfrom the reaction mixture with the biological sample is less than theread-out from the reaction mixture without the biological sample,formation of the complex is decreased. The amount of complex formed isinversely proportional to the presence of the anti-thyroid peroxidaseantibody in the biological sample.

In some embodiments, the methods can comprise a “competition”immunoassay or an “inhibition” immunoassay, an exemplary reaction schemefor which is illustrated in FIG. 4. As used herein, a “competition”immunoassay refers to an immunoassay in which two or more bindingmolecules compete for binding to a target molecule. The bindingmolecules can be antibodies, for example, and the target molecule can bean antigen. The binding molecules can compete, for example, for bindingto the same epitope(s) on the antigen. An “inhibition” immunoassayrefers to an immunoassay in which one or more binding molecules inhibitbinding of one or more other binding molecules to a target molecule. Thebinding molecules can be antibodies, for example, and the targetmolecule can be an antigen. Inhibition can be caused by steric hindranceor other known mechanisms of binding inhibition. Whether the claimedassays are classified as a “competition” or “inhibition” immunoassaywill depend, for example, on the rTPO epitope recognized by theanti-thyroid peroxidase antibody in the biological sample and thelabeled anti-TPO antibody. The claimed methods encompass bothcompetition and inhibition immunoassays, and thus the terms“competition” and “inhibition” are not intended to limit the scope ofthe claimed methods.

In some embodiments of the competition/inhibition immunoassay, thelabeled anti-TPO antibody and the rTPO can be in pre-formed immunecomplex prior to their addition to the reaction mixture. In someembodiments, the unlabeled anti-TPO antibody and solid support can be inpre-formed complex prior to their addition to the reaction mixture. Insome embodiments, the labeled anti-TPO antibody, the rTPO, the unlabeledanti-TPO antibody, and the solid support are not in a pre-formed complexprior to their addition to the reaction mixture. In yet otherembodiments, the labeled anti-TPO antibody, the rTPO, the unlabeledanti-TPO antibody, and the solid support are all present in a pre-formedcomplex prior to their addition to the reaction mixture. Whether thevarious components are in one or more pre-formed complexes prior totheir addition to the reaction mixture will depend, in part, on thedissociation constants (K_(D)) of the labeled and unlabeled antibodiesand the rTPO. The disclosed methods are not limited by the type orextent of complex formation added to the reaction mixture. It is to beunderstood that the biological sample, the labeled anti-TPO antibody,the rTPO, the unlabeled anti-TPO antibody, and the solid support can beadded to the reaction mixture in any order.

FIG. 4 discloses one exemplary embodiment of the competition/inhibitionimmunoassays described herein, in which a labeled anti-TPO antibody 110and an rTPO 120 are incubated with a solid support having an unlabeledanti-TPO antibody bound thereto 130. In the absence of the anti-TPOantibody from the biological sample 10, the labeled anti-TPO antibody110 will bind to the rTPO 120 and in turn bind to or interact with thesolid support having unlabeled anti-TPO antibody bound thereto 130,thereby forming a complex 140 comprising the labeled anti-TPO antibody110, the rTPO 120, and the solid support. Thus, in the absence of theanti-TPO antibody from the biological sample 10, the labeled anti-TPOantibody 110 will be coupled to the solid support, isolation of whichwill result in isolation of the labeled anti-TPO antibody 110. When theanti-TPO antibody 10 is present in the biological sample, the anti-TPOantibody 10 will compete with or otherwise inhibit the labeled anti-TPOantibody 110 for binding to the rTPO 120 or will compete with orotherwise inhibit the unlabeled anti-TPO antibody bound to the solidsupport 130 for binding to the rTPO 120, thereby preventing the complexfrom forming or displacing the labeled anti-TPO antibody from thecomplex 140. Thus, in the presence of anti-TPO antibody from thebiological sample 10, the presence of complex 140 comprising the labeledanti-TPO antibody 110, the rTPO 120, and the solid support is decreased.

The labeled anti-TPO antibody 110, the unlabeled rTPO 120, and theunlabeled anti-TPO antibody bound to the solid support 130 can beincubated in a reaction mixture for about 1 minute, about 2 minutes,about 3 minutes, about 4 minutes, about 5 minutes, or less than about 10minutes. The biological sample known to have, or suspected of having, ananti-TPO antibody 10 can be added and incubated in the reaction mixturefor about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes,about 5 minutes, or less than about 10 minutes. In some embodiments, theincubating steps are performed in a total of about 10 minutes to about20 minutes. The subsequent detecting can be performed in less than about5 minutes. It is to be understood that the amount of time needed for theassay may vary based upon several factors including the level of theanti-TPO antibody(ies) in the biological sample and the affinity of theanti-TPO antibody(ies) for the rTPO. Thus, the disclosed methods can beperformed for any suitable amount of time.

The unlabeled anti-TPO antibody can be directly or indirectly coupled tothe solid support. Suitable techniques for directly coupling theunlabeled anti-TPO antibody to the solid support include, for example,covalent attachment, adsorption, noncovalent interaction, orcombinations thereof. Suitable means for indirectly coupling theunlabeled anti-TPO antibody to the solid support include, for example,linking through a peptide, a protein, an antibody, a linker, or acombination thereof. In some embodiments, the unlabeled anti-TPOantibody can be indirectly coupled to the solid support throughstreptavidin and biotin. For example, the unlabeled anti-TPO antibodycan be biotinylated and the solid support can comprise streptavidin. Thelabeled and/or the unlabeled anti-TPO antibody can be any knowncommercially available anti-TPO antibody.

Exemplary solid supports include, but are not limited to, a columnmatrix material, a culture plate, a tube, a dish, a flask, a microtiterplate, a bead/particle, heat-killed formalin- (or otherchemically)-fixed prokaryotic or eukaryotic cells, microscope slides,ACLAR® Film, or any other optically transparent polymer, or acombination thereof. The solid support can be fully or partiallycomposed of plastic, cellulose, cellulose derivatives, nitrocellulose,glass, fiberglass, latex, or a combination thereof. In some embodiments,the solid support comprises a magnetic bead/particle. In someembodiments, the magnetic bead/particle is a paramagnetic particle(PMP). In some embodiments, the magnetic bead/particle is a latexmagnetic particle (LMP).

The label can be any suitable label known to those skilled in the art tobe useful for creating a detectable signal. Suitable detectable labelsinclude, but are not limited to, enzyme conjugates (e.g., horseradishperoxidase (HRP), alkaline phosphatase, glucose oxidase, andβ-galactosidase), fluorescent probes, radioactive isotopes,chemiluminescent compounds, bioluminescent compounds, or combinationthereof. In some embodiments, the label is an AE or an analog thereof.Suitable AE analogs include: dimethyl acridinium ester (DMAE),N-sulfopropyl dimethyl acridinium ester (NSP-DMAE), high quantum yieldacridinium ester (HQYAE), Zwitterionic acridinium ester (ZAE),N-sulfopropyl-2-isopropoxy dimethyl acridinium ester (Iso-Di-ZAE),trisulfopropyl acridinium ester (TSP-AE), or N-sulfopropyl dimethylacridinium ester with hexa(ethylene)glycol linker (HEG-GLU-AE). In someembodiments, the labeled anti-TPO antibody is anti-TPO IgG HEG-GLU-AE.The anti-rTPO IgG HEG-GLU-AE can be present at about 50 ng/ml to about 2μg/ml. In some embodiments, the anti-rTPO IgG HEG-GLU-AE can be presentat about 420 ng/ml.

The solid support having unlabeled anti-TPO antibody bound theretoand/or the unlabeled rTPO and/or the labeled anti-TPO antibody can bepresent in a buffer comprising, for example, phosphate buffer, NaCl,EDTA, pluronic F-127, sodium azide, sorbitol, sulfhydryl modified bovineserum, or any combinations, variations, or equivalents thereof. In oneembodiment, the buffer comprises about 100 mM phosphate buffer, about400 mM NaCl, about 1.9 g/L EDTA, about 0.2% (v/v) pluronic F-127, about0.9 g/L sodium azide, about 10% sorbitol, and about 10 g/L sulfhydrylmodified bovine serum albumin.

Further disclosed are methods of diagnosing a thyroid disease in asubject, the methods comprising incubating a biological sample from thesubject with a solid support, an unlabeled recombinant cynomolgus monkeythyroid peroxidase (rTPO), and a labeled cynomolgus monkey rTPO. In thepresence of an anti-thyroid peroxidase antibody in the biologicalsample, a complex comprising the labeled cynomolgus monkey rTPO, theunlabeled cynomolgus monkey rTPO, and the solid support is formed. Themethod further comprises diagnosing the subject with the thyroid diseaseif the complex is formed.

Methods of diagnosing a thyroid disease in a subject comprisingincubating a biological sample from the subject with a solid support, acynomolgus monkey rTPO, and an anti-human secondary antibody are alsoprovided. In the presence of an anti-thyroid peroxidase antibody in thebiological sample, a complex comprising the solid support, thecynomolgus monkey rTPO, and the anti-human secondary antibody is formed.The method further comprises diagnosing the subject with the thyroiddisease if the complex is formed.

Also disclosed are methods of diagnosing a thyroid disease in a subject,the methods comprising incubating a biological sample from a subjectwith a solid support, an unlabeled anti-TPO antibody, a cynomolgusmonkey TPO, and a labeled anti-TPO antibody, and diagnosing the thyroiddisease if the formation of a complex comprising the solid support, theunlabeled anti-TPO antibody, the cynomolgus monkey rTPO, and the labeledanti-TPO antibody is decreased. The presence of an anti-thyroidperoxidase antibody in the biological sample decreases formation of acomplex comprising the solid support, the unlabeled anti-TPO antibody,the cynomolgus monkey rTPO, and the labeled anti-TPO antibody. Thelabeled and/or the unlabeled anti-TPO antibody can be any known orcommercially available anti-TPO antibody.

The methods of diagnosing can further comprise a step of detecting thecomplex. For example, detecting the complex may comprise taking aread-out of a signal from the label, wherein the intensity of the signalfrom the label indicates the amount of label present in the assay.

The thyroid disease can be an autoimmune disorder. In some embodiments,the autoimmune disorder is Hashimoto's thyroiditis or Graves' disease.

Similar to the methods of detecting described above, in the methods ofdiagnosing, the unlabeled rTPO, the unlabeled anti-human secondaryantibody, or the unlabeled anti-TPO antibody can be directly orindirectly linked/coupled to the solid support. Suitable techniques fordirect linking/coupling include, for example, covalent attachment,adsorption, noncovalent interaction, or combinations thereof. In someembodiments, the direct linking/coupling can be achieved byglutaraldehyde fixation, N-hydroxysuccinimide (NETS) chemistry, or1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) NETS chemistry.Suitable means for indirect linking/coupling include, for example,linking/coupling through a peptide, a protein, an antibody, a linker, ora combination thereof. In some embodiments, the indirectlinking/coupling to the solid support is via streptavidin and biotin.For example, the unlabeled rTPO, the unlabeled anti-human secondaryantibody, or the unlabeled anti-TPO antibody can be biotinylated and thesolid support can comprise streptavidin.

Exemplary solid supports include, but are not limited to, a columnmatrix material, a culture plate, a tube, a dish, a flask, a microtiterplate, a bead/particle, heat-killed formalin- (or otherchemically)-fixed prokaryotic or eukaryotic cells, microscope slides,ACLAR® Film, or any other optically transparent polymer, or acombination thereof. The solid support can be fully or partiallycomposed of plastic, cellulose, cellulose derivatives, nitrocellulose,glass, fiberglass, latex, or a combination thereof. In some embodiments,the solid support comprises a magnetic bead/particle. In someembodiments, the magnetic bead/particle is a paramagnetic particle(PMP). In some embodiments, the magnetic bead/particle is a latexmagnetic particle (LMP).

The label can be any suitable label known to those skilled in the art tobe useful for creating a detectable signal. Suitable detectable labelsinclude, but are not limited to, enzyme conjugates (e.g., horseradishperoxidase (HRP), alkaline phosphatase, glucose oxidase, andβ-galactosidase), fluorescent probes, radioactive isotopes,chemiluminescent compounds, bioluminescent compounds, or combinationthereof. In some embodiments, the label is an AE or an analog thereof.Suitable AE analogs include: dimethyl acridinium ester (DMAE),N-sulfopropyl dimethyl acridinium ester (NSP-DMAE), high quantum yieldacridinium ester (HQYAE), Zwitterionic acridinium ester (ZAE),N-sulfopropyl-2-isopropoxy dimethyl acridinium ester (Iso-Di-ZAE),trisulfopropyl acridinium ester (TSP-AE), or N-sulfopropyl dimethylacridinium ester with hexa(ethylene)glycol linker (HEG-GLU-AE). In someembodiments, the labeled cynomolgus monkey rTPO comprises rTPO-NSP-DMAE.The rTPO-NSP-DMAE can be present at about 50 ng/ml to about 2 μg/ml. Insome embodiments, the rTPO-NSP-DMAE can be present at about 220 ng/ml.In some embodiments, the labeled anti-TPO antibody is anti-rTPO IgGHEG-GLU-AE. The anti-rTPO IgG HEG-GLU-AE can be present at about 50ng/ml to about 2 μg/ml. In some embodiments, the anti-rTPO IgGHEG-GLU-AE can be present at about 420 ng/ml.

Suitable times for performing the methods of diagnosing an autoimmunedisease include those provided above for the various methods ofdetecting an antibody.

The methods can further comprise determining a level of the anti-thyroidperoxidase antibody in the biological sample of the subject. In someembodiments, the level of the anti-thyroid peroxidase antibody in thebiological sample of the subject is directly proportional to the levelof the complex detected. Thus, the level of anti-TPO antibody can bedetermined by determining the level of solid support coupled to or incomplex with the labeled component (i.e. the labeled rTPO or antibody).Determining the level of solid support coupled to or in complex with thelabeled component can be performed, for example, by measuring the signalfrom the complex. Similarly, in embodiments wherein the method is usedto diagnose a thyroid disease in a subject, the solid support coupled toor in complex with the labeled component is detected by measuring thesignal or absence thereof from the labeled component linked to the solidsupport.

The disclosed methods can be performed manually or can be automated. Forexample, the disclosed methods can be performed using an ADVIA CENTAUR®Immunoassay System or an ATELLICA™ system.

In some embodiments, the anti-TPO antibody is a patient anti-TPOantibody. In some embodiments, the anti-TPO antibody is an autoantibody.

Suitable biological samples for detecting the anti-TPO antibody includeany biological sample from a subject that contains, or is suspected ofcontaining, the anti-TPO antibody including, but not limited to, serum,plasma, whole blood, saliva, urine, semen, perspiration, tears, and bodytissues.

The immunoassays disclosed herein employ a recombinant thyroidperoxidase (rTPO). In some embodiments, the rTPO is from cynomolgusmonkey. In some embodiments, the rTPO comprises the amino acid sequenceset forth in SEQ ID NO: 1 (Table 1). In some embodiments, the rTPOfurther comprises an epitope tag. The epitope tag can be at theN-terminus or at the C-terminus of the rTPO. The epitope tag can be anysuitable tag known to persons skilled in the art including, but notlimited to, a 6-histidine tag, a hemagglutinin tag, aglutathione-S-transferase, a maltose binding protein, or a chitinbinding protein. In some embodiments, the rTPO comprises a C-terminal6-histidine tag.

TABLE 1 Macaca fascicularis rTPO amino acid sequence (SEQ ID NO: 1)SEQ ID NO: 1 ADPGYLLECT EAFFPFISRG KELLWGKPEE SRVAGILEES KRLVDTAMYATMQRNLKKRE ILSPHQLLSF SKLPEPTSGE IARAAEIMET SIQAMKRKVNLKIQQSQHPT DALSEDLLSI IANMSGCLPY MLPPKCPNTC LANKYRPITGACNNRDHPRW GASNTALARW LPPVYEDGFS QPRGWNPSIL HNGFPLPPVREVTRHVIQVS NEVVTDDDRY SDLLMAWGQY IDHDIAFTPQ STSKAAFRGGADCQVTCENQ NPCFPIQLPE EARPAAGTAC LPFYRSSAAC GTGDQGALFGNLSTANPRQQ MNGLTSFLDA STVYGSSPAL ERQLRNWTSA EGLLRVHARLRDSGRAYLPF APPRAPAACA PEPGIPGETR GPCFLAGDGR ASEVPSLTALHTLWLREHNR LAAALKALNA HWSADAVYQE ARKVVGALHQ IITLRDYVPRILGPEAFQQY VGPYEGYDSA ANPTVSNVFS TAAFRFGHAT IHPLVRRLDAGFQEHPGLPG LWLHETFFSP WTLLHGGGLD PLIRGLLARP AKLQVQDQLMNEELTERLFV LSNSSTLDLA SINLQRGRDH GLPGYNEWRE FCGLPRLETPADLSTAIASR SVADKILDLY KHPDNIDVWL GGLAENFLPR ARTGPLFACLIGKQMKALRD GDWFWWENSH VFTDAQRHEL EKHSLSRVIC DNTGLTRVPVDAFRVGKFPE DFESCDSIPG MNLEAWRETF PQDDKCGFPE SVENGDFVHCEESGRRVLVY SCRHGYELQG HEQLTCTQEG WDFQPPLCKD VNECADGAHPPCHASARCRN TKGGFQCLCA DPYELGDDGR TCVDSGRLPR

Further disclosed herein are kits. In some embodiments, the kits cancomprise a solid support, an unlabeled cynomolgus monkey rTPO, and alabeled cynomolgus monkey rTPO.

The kits can comprise a solid support, a cynomolgus monkey rTPO, and ananti-human secondary antibody. In some embodiments, the cynomolgusmonkey rTPO comprises a label or the anti-human secondary antibodycomprises a label.

The kits can comprise a solid support, a cynomolgus monkey TPO and ananti-TPO antibody. In some embodiments, a portion of the anti-TPOantibodies are unlabeled and a portion of the anti-TPO antibodies arelabeled.

Suitable solid supports and labels for any of the kits disclosed hereininclude those solid supports and labels disclosed for the methods above.

EXAMPLES

The following examples are provided to further describe some of theembodiments disclosed herein. The examples are intended to illustrate,not to limit, the disclosed embodiments.

rTPO Cloning

Monkey thyroid tissue was obtained from cynomolgus monkey and mRNA wasisolated and purified using Trizol/Chloroform extraction (Invitrogen,catalogue #15596026). A cDNA library was prepared using InvitrogenThermo-Script RT-PCR kit.

Using PCR, a soluble form of TPO was amplified from the cDNA library andthe PCR product was purified using a Qiagen PCR purification kit(QIAquick PCR Purification Kit, catalogue #28104). The purified PCRproduct was digested with XbaI (New England Biolabs cat # R0145S) andNotI (New England Biolabs cat # R0189S) restriction endonucleases andgel-extracted. The gel-extracted TPO was ligated into a baculovirusexpression vector. The vector was transformed in competent cells andplated on ampicillin-selected plates.

Resulting colonies were PCR-screened and positive clones were grown forplasmid generation and purification. Several plasmid clones weresequenced using a Beckman CEQ 8000 sequencer; clones containing TPO wereverified. All PCR and sequencing primers were synthesized using an ABIExpedite 8909 DNA Synthesizer. The cDNA encodes a cynomolgus monkey rTPOhaving the amino acid sequence of SEQ ID NO. 1 (Table 1).

Transient Transfection to Yield rTPO Baculoviruses

Baculoviral DNA was combined with a transfer plasmid comprising the rTPOsequence and incubated with Grace's media and transfection reagent(Invitrogen, Bac-N-Blue Transfection Kit, catalogue # K855-01). Aftertransfection of Spodoptra frugiperda (Sf) cells, cell culturesupernatants were subsequently screened for recombinant viruses andselected recombinants were purified, amplified and sequenced to confirmthe identity of rTPO.

rTPO Purification

Purified recombinant baculovirus containing the rTPO was used to infectcultures of Trichoplusia ni PRO (Tni PRO) in ESF921 cell culture medium.Transduced rTPO was secreted into the cell culture supernatant.

rTPO was isolated from the supernatant using Chelating Sepharose FastFlow immobilized metal (Nickel) affinity chromatography followed by sizeexclusion chromatography. Briefly, the chelating sepharose gel (GEHealthcare Catalog No. 17-0575-01) was charged with 100 mM NiSO₄. rTPOin the culture supernatant was allowed to bind to the gel for 1.5 hours.Non-specific proteins were removed by washing the gel with 0.5 M SodiumChloride, 0.05 M Tris pH 8.0, 10 mM Imidazole. rTPO was then eluted with0.5 M Sodium Chloride, 0.05 M Tris pH 8.0, 0.1 M Imidazole. The elutionfraction was then subjected to S-200 size exclusion chromatography onAKTA Prime Plus liquid chromatography system, and fractions containingrTPO were pooled.

The final pool containing pure rTPO was analyzed by SDS/PAGE, followedby Coomassie Blue staining (FIG. 5, panel A) or western blotting (FIG.5, panel B). rTPO was electrophoresed on 4-20% Tris-Glycine gels undernon-reducing conditions for about 30-40 minutes at 200 V. For westernblot analysis, the proteins were transferred onto nitrocellulosemembrane and were probed with a mouse anti-TPO monoclonal antibody forone hour, followed by an horse radish peroxidase (HRP) conjugated, goatanti-mouse IgG polyclonal antibody (Millipore, lot 17011271) for 30minutes; blots were developed with substrate (Surmodics BCIB) for about5 minutes.

Glycosylation

The extent of rTPO glycosylation was determined by ion exchangechromatography, matrix-assisted LASER desorption time-of-flight(“MALDI-TOF”) mass spectrometry, and isoelectric focusing. In each ofthe ion exchange chromatography, MALDI-TOF, and isoelectric focusingexperiments, two protein species were identified, which appeared to bethe result of glycosylation. As shown in FIG. 6, the major band had anaverage pI 6.2 and the minor band had an average pI 5.2.

To further characterize potential lot-to-lot glycan diversity in rTPO,three different lots of rTPO were each digested with one of thefollowing enzymes: Protein Deglycosylation Mix II (including PNGase F,O-Glycosidase, α2-3,6,8,9 Neuraminidase A, β1-4 Galactosidase S, andβ-N-acetylhexosaminidase_(f)), EndoH, α2-3, 6, 8, 9 Neuraminidase,O-Glycosidase, or PNGaseF (New England Biolabs). Digested rTPO wassubsequently analyzed by MALDI-TOF analysis using the Shimadzu AXIMAConfidence MALDI-TOF Mass Spectrometer. Enzyme digestion conditions foreach reaction are summarized in Table 2 below. After 37° C. overnightincubation, samples were collected, cooled to room temperature, andde-salted. A 10 kDa MWCO centrifugal filter (Millipore; ref#UFC501024)was washed with 250 μl diH₂O and spun for 10 minutes at 14,000 rpm.Samples were de-salted by adding digested sample to washed centrifugalfilter, spinning at 14,000 rpm for 10 minutes, and decanting theflow-through. After each spin, 250 μl diH₂O was aliquoted into thecentrifugal filter and spun again at 14,000 rpm for 10 minutes for atotal of three washes.

TABLE 2 Enzyme Digestion Conditions. Deglycosylation Mix EndoHNeuraminidase O-glycosidase PNGaseF Catalog #P6044S Catalog #P0702SCatalog #P0720S Catalog #P0733S Catalog #P0704S 110 μL rTPO 45 μL rTPO20 μL rTPO 45 μL rTPO 45 μL rTPO 40 μL H2O 5 μL 10X 160 μL H2O 5 μL 10X5 μL 10X glycosylation glycosylation glycosylation denaturationdenaturation denaturation Buffer Buffer Buffer 5 μL Place mixture in 2μL 10X Place mixture in Place mixture in Deglycosylation fully boilingH₂O GlycoBuffer 1 fully boiling H₂O fully boiling H₂O Mix Buffer 2 for10 minutes for 10 minutes for 10 minutes Incubate at 75° C. 10 μL 20 μL10 μL 10 μL for 10 minutes, cool GlycoBuffer 3 Neuraminidase GlycoBuffer2 GlycoBuffer 2 to RT 10X 10X 10X Incubate at 25° C. for 15 μL EndoH 10μL 5 μL PNGaseF 30 mins Neuraminidase 25 μL H₂O 15 μL O- 35 μL H₂Oglycosidase 15 μL H₂O All reactions were incubated overnight in a 37° C.warm water bath.

After 37° C. overnight incubation, samples were collected, cooled toroom temperature, and de-salted. A 10 kDa MWCO centrifugal filter(Millipore; ref # UFC501024) was washed with 250 μL diH₂O and spun for10 minutes at 14,000 rpm. Samples were de-salted by adding digestedsample to washed centrifugal filter, spinning at 14,000 rpm for 10minutes, and decanting the flow-through. After each spin, 250 μL diH₂Owas aliquoted into the centrifugal filter and spun again at 14,000 rpmfor 10 minutes for a total of three washes.

All samples were desalted and reconstituted directly with a sinapinicacid (Sigma-Aldrich 49508) trifluoroacetic acid (Alfa-Aesar 44630)matrix solution. The sample to matrix ratio was optimized for eachsample, with optimal ratios outlined in Table 3. A 2 μl aliquot fromeach sample was blotted in triplicate on the MALDI-TOF sample plate. Thesamples were thoroughly air dried, placed in the MALDI-TOF samplechamber, and measured on a Shimadzu Axima Confidence MALDI-TOF withlaser power set at 65. The MALDI-TOF mass spectrometer was calibratedusing ProteoMass Albumin Standard (Sigma; Part # A8471).

TABLE 3 Optimized sample-to-matrix ratios for enzyme digests. SampleRatio (Sample-to-Matrix) De-glycosylation Mix 1:2 EndoH 1:1Neuraminidase 1:2 O-glycosidase 1:2 PNGaseF 1:1 Undigested rTPO 1:2

MALDI-TOF analyses of three lots of rTPO showed slight lot-to-lotdifferences in glycosylation (data not shown). Digestion withDe-glycosylation Mix showed the most pronounced molecular weight shift;this enzyme was chosen for de-glycosylation and subsequent conjugationfor functional testing on ADVIA CENTAUR® XP immunoassay (SiemensHealthcare).

rTPO Immunoassay Characterization

N-hydroxysuccinimide (NETS) chemistry was used to prepare native humanTPO (Fitzgerald 80-1382) labeled with NSP-DMAE and recombinantcynomolgus monkey TPO labeled with NSP-DMAE. These labeled proteins werepaired with biotinylated anti-human IgG coupled to streptavidin latex orpara magnetic particles in 100 mM phosphate buffer, 400 mM NaCl, 1.9 g/LEDTA, 0.2% (v/v) pluronic F-127, 0.9 g/L sodium azide, 10% sorbitol, and10 g/L sulfhydryl modified bovine serum albumin and tested in a 7.5minute one-pass (time to final result (TTFR): 18 minutes) and/or 20/20minute two-pass (TTFR: 60 minutes) formats using an ADVIA CENTAUR®system (Siemens Healthcare). In the 7.5 minute one-pass immunoassays,the sample was added at time zero. Human TPO-NSP-DMAE or rTPO-NSP-DMAEwas added at 4.75 minutes, followed by reagent containing latex magneticparticles at 7.5 minutes. Magnetic separation was initiated at 13.0minutes to isolate the latex magnetic particles. In the 20/20 two-passimmunoassays, sample was added at time zero and first-pass humanTPO-NSP-DMAE or rTPO-NSP-DMAE was added at 6 minutes, followed byfirst-pass magnetic separation at 24.0 minutes. Second-pass humanTPO-NSP-DMAE or rTPO-NSP-DMAE was added at 35.0 minutes, and second-passmagnetic separation was initiated at 52.75 minutes. In both the one-passand two-pass formats, the magnetic particles were prepared by washingwith phosphate buffered saline three times and resuspending in 100 mMphosphate buffer, 400 mM NaCl, 1.9 g/L EDTA, 0.2% (v/v) pluronic F-127,0.9 g/L sodium azide, 10% sorbitol, and 10 g/L sulfhydryl modifiedbovine serum albumin. In the cuvette the particles were magneticallyseparated and resuspended in either water or tween-20 PBS according tothe Centaur platform cycle parameters. Siemens aTPO Master CurveMaterial (Siemens 10630890) were run as samples. As shown in FIG. 11,recombinant TPO gave a higher signal-to-noise ratio compared to nativeTPO using both 7.5 minute one-pass and 20/20 minute two-pass formats onthe ADVIA CENTAUR® XP.

Anti-TPO Antibody Immunoassays Antigen Bridge Format

The rTPO antigen bridge immunoassay used biotinylated rTPO directlycoupled to streptavidin-coated latex magnetic particles in 0.6 mg/mL in100 mM phosphate buffer, 400 mM NaCl, 1.9 g/L EDTA, 0.2% (v/v) pluronicF-127, 0.9 g/L sodium azide, 10% sorbitol, and 10 g/L sulfhydrylmodified bovine serum albumin, and rTPO coupled to TSP-AE (30 ng/mLrTPO-TSP-AE in 100 mM phosphate buffer, 400 mM NaCl, 1.9 g/L EDTA, 0.2%(v/v) pluronic F-127, 0.9 g/L sodium azide, 10% sorbitol, and 10 g/Lsulfhydryl modified bovine serum albumin). The rTPO antigen bridgeformat was run as a 7.5 minute one-pass (TTFR: 18 minutes) assay andrequired a 1:6 onboard, sample predilution. On an ADVIA CENTAUR® XP, 20μL of diluted sample was added to a second cuvette and incubated for4.75 minutes. 100 of rTPO-TSP-AE was added and incubated for 2.75minutes. 100 μL of rTPO/latex magnetic particles was then added andincubated for 2.75 minutes. Magnetic separation was initiated at 13.0minutes. The magnetic particles were washed with phosphate bufferedsaline three times, and finally resuspended to their originalconcentration with 100 mM phosphate buffer, 400 mM NaCl, 1.9 g/L EDTA,0.2% (v/v) pluronic F-127, 0.9 g/L sodium azide, 10% sorbitol, and 10g/L sulfhydryl modified bovine serum albumin. In the cuvette theparticles were magnetically separated and resuspended in either water ortween-20 PBS according to the Centaur platform cycle parameters. Anexemplary schematic of the Antigen Bridge assay format is shown inFIG. 1. The performance of an optimized rTPO antigen bridge immunoassay(optimized to minimize background, increase signal-to-noise and limitany prozone effect) is provided in FIG. 12A.

IgG Class Capture Format

The human IgG class capture immunoassay used anti-human IgG monoclonalantibody directly coupled to paramagnetic particles by glutaraldehydefixation and rTPO coupled to NSP-DMAE (0.34 mg/mL anti-humanIgG/paramagnetic particles and 220 ng/mL rTPO-NSP-DMAE, both in 100 mMphosphate buffer, 400 mM NaCl, 1.9 g/L EDTA, 0.2% (v/v) pluronic F-127,0.9 g/L sodium azide, 10% sorbitol, and 10 g/L sulfhydryl modifiedbovine serum albumin). The human IgG class capture format was run as a7.5 minute one-pass (TTFR: 18 minutes) assay and required a 1:10onboard, sample predilution. On an ADVIA CENTAUR® XP, 20 μL of dilutedsample was added to a second cuvette and incubated for 4.75 minutes. 100μL of rTPO-NSP-DMAE was added and incubated for 2.75 minutes. 200 μL ofanti-human IgG/paramagnetic particles was added and incubated for 2.75minutes. Magnetic particles were separated and washed as describedabove. An exemplary schematic of the IgG Class Capture assay format isdepicted in FIG. 2. The performance of an optimized human IgG classcapture immunoassay (optimized to minimize background, increasesignal-to-noise and limit any prozone effect) is provided in FIG. 12B.

rTPO Capture Format

The rTPO capture immunoassay used rTPO directly coupled to latexmagnetic particles using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC) NHS chemistry and a monoclonal anti-human IgG antibody coupled toNSP-DMAE (0.4 mg/mL rTPO/latex magnetic particles and 1.6 μg/mLanti-human IgG antibody-NSP-DMAE, both in 100 mM phosphate buffer, 400mM NaCl, 1.9 g/L EDTA, 0.2% (v/v) pluronic F-127, 0.9 g/L sodium azide,10% sorbitol, and 10 g/L sulfhydryl modified bovine serum albumin). TherTPO capture format was run as a 7.5 minute one-pass (TTFR: 18 minutes)assay and required a 1:10 onboard, sample predilution. On an ADVIACENTAUR® XP, 20 μL of diluted sample was added to a second cuvette. 100μL of anti-human IgG antibody-NSP-DMAE was added and incubated for 2.75minutes. 100 μL of rTPO/latex magnetic particles was added and incubatedfor 2.75 minutes. Magnetic particles were separated and washed asdescribed above. An exemplary schematic of the rTPO Capture assay formatis depicted in FIG. 3. The performance of an optimized rTPO captureimmunoassay (optimized to minimize background, increase signal-to-noiseand limit any prozone effect) is provided in FIG. 12C.

Competition/Inhibition Format

The competition/inhibition immunoassay used a biotinylated monoclonalmouse anti-rTPO IgG antibody coupled to streptavidin-coated latexmagnetic particles and monoclonal mouse anti-rTPO IgG antibody coupledto HEG-GLU-AE in immune complex with unlabeled rTPO (respectively, 0.3mg/mL mouse-anti-rTPO/latex magnetic particles and 420 ng/mLrTPO:anti-rTPO IgG HEG-GLU-AE, both in 100 mM phosphate buffer, 400 mMNaCl, 1.9 g/L EDTA, 0.2% (v/v) pluronic F-127, 0.9 g/L sodium azide, 10%sorbitol, and 10 g/L sulfhydryl modified bovine serum albumin). Thecompetition/inhibition format was run as a 7.5 minute one-pass (TTFR: 18minutes) assay. On an ADVIA CENTAUR® XP, 20 μL of undiluted sample wasadded to a cuvette and incubated for 4.75 minutes. 75 μL ofrTPO:anti-rTPO IgG HEG-GLU-AE and 150 μL of mouse-anti-rTPO/latexmagnetic particles were added and incubated for 2.75 minutes each.Magnetic particles were separated and washed as described above. Anexemplary schematic of the competition/inhibition assay format is shownin FIG. 4. The performance of an optimized competition/inhibitionimmunoassay (optimized to minimize background, increase signal-to-noiseand limit any prozone effect) is provided in FIG. 12D.

Effect of rTPO Glycosylation on Immunoassay Performance

To analyze the effect of recombinant rTPO glycosylation on immunoassayperformance, immunoassays were run using the Antigen Bridge and IgGClass Capture (with labeled rTPO detection) formats. Table 4 outlinesimmunoassay preparation for each format. Immunoassays were run intriplicate on an ADVIA CENTAUR® XP. Samples were prepared using anti-TPOantibody-Positive Plasma spiked into PBS Buffer targeting the followingdoses: 0, 12.5, 25, 50, 100, 200, 300, 400, 625, 20,000 IU/mL.

In the Antigen Bridge assay format, 30 μL of sample was added to acuvette, followed by 180 μL diluent (100 mM phosphate buffer, 400 mMNaCl, 1.9 g/L EDTA, 0.2% (v/v) pluronic F-127, 0.9 g/L sodium azide, 10%sorbitol, 10 g/L sulfhydryl modified bovine serum albumin), and thediluted sample was incubated for 4.75 minutes. 30 μL of the dilutedsample was added to 100 μL diluent containing paramagnetic particles,and incubated for 2.75 minutes. 100 μL of the diluent containing labeledrTPO was added to the reaction and incubated for 6.5 minutes. Theimmunoassay was tested (results shown in FIG. 7A) with node-glycosylation (“Control”), de-glycosylated labeled rTPO(“Glycosylated SP/Deglycosylated LR”), de-glycosylated unlabeled rTPObound to the solid support (“Deglycosylated SP/Glycosylated LR”), andboth de-glycosylated labeled rTPO and unlabeled rTPO bound to the solidsupport (“Deglycosylated SP & LR”).

In the IgG Class Capture assay format, 30 μL of sample was added to acuvette, followed by 180 μL diluent (100 mM phosphate buffer, 400 mMNaCl, 1.9 g/L EDTA, 0.2% (v/v) pluronic F-127, 0.9 g/L sodium azide, 10%sorbitol, 10 g/L sulfhydryl modified bovine serum albumin), and thediluted sample was incubated for 4.75 minutes. 30 μL of the dilutedsample was added to 100 μL diluent containing labeled rTPO, andincubated for 2.75 minutes. 100 μL of the diluent containing anti-humanIgG Fc monoclonal antibody coupled to paramagnetic particles was addedto the reaction and incubated for 6.5 minutes. The immunoassay wastested with non-deglycosylated labeled rTPO (control) andde-glycosylated labeled rTPO (FIG. 7B).

TABLE 4 Immunoassay preparation Solid Support (bound to [Solid [Labeledmagnetic Support] Labeled rTPO] particles) (mg/mL) rTPO (ng/mL) AntigenrTPO: Sulfo- 0.6  rTPO: NSP- 120 Bridge NHS-LC- mg/mL DMAE-Z- ng/mLBiotin 30X NHS 20X IgG Class Anti-human 0.34 rTPO: NSP- 220 Capture w/rTPO IgG, 8D6 mg/mL DMAE-Z- ng/mL Detection NHS 20X

A functional assessment of rTPO, based on glycosylation, revealed thatdifferences in rTPO glycosylation result in subtle variation inimmunoreactivity (as measured by ADVIA CENTAUR® XP) against an anti-TPOantibody-positive patient pool (FIG. 7A and FIG. 7B).

The immunoassays demonstrated low background and a wide dynamic rangefor effective aTPO detection and quantification.

rTPO Immune Complex Characterization

FPLC size exclusion chromatography was employed to verify the in vitroformation of a recombinant TPO—anti-TPO autoantibody complex and toconfirm that the anti-TPO autoantibody, rather than some other componentwithin the patient sample, was interacting with the recombinant TPO.

rTPOAE baseline chromatogram: rTPO labeled with N-sulfopropyl dimethylacridinium ester (NSP-DMAE) (5 molar excess) was diluted to get ˜5million RLU (1:600 dilution) using fractionation buffer; 50 mM SodiumPhosphate, 150 mM sodium Chloride, 0.5% Tween 20, and 0.1% Sodium Azideat pH 7.4. This sample was analyzed by AKTA pure system with Superdex200 HiLoad 16/600 column using the fractionation buffer, 5004, injectionand 1 ml/minute elution; 180 fractions (1 ml each) were collected andrun on Berthold AutoLumat system to generate a baseline RLUchromatogram. The labeled rTPO was represented by a single peak at MW90-100 KDa (fractions 66-76) in the RLU chromatogram in FIG. 8 (Note:“rTPOAE” refers to rTPO labeled with an acridinium ester analog orvariant, e.g., NSP-DMAE in the present example).

In vitro immune complex chromatogram: A high anti-TPO antibody patientsample pool (concentration 60,000 U/mL) was diluted to 1,500 U/mL withthe fractionation buffer. One part of the diluted sample was mixed with19 parts of the diluted (1:600) rTPO labeled with dimethyl acridiniumester (NSP-DMAE). The sample mixture was analyzed using the same columnand conditions as above. The RLU chromatogram of the mixture overlaidwith the baseline chromatogram in FIG. 8 (“rTPOAE spiker”) shows ahigher molecular weight immune complex peak (fractions 45-55) along withthe unbound labeled rTPO peak (Fractions 66-76).

MALDI-TOF was used to evaluate the components of the immune complex.

Sample preparation: A high anti-TPO antibody patient pool (concentration20,000 IU/mL) was diluted to 2,700 IU/mL with the fractionation buffer.The diluted sample was mixed with rTPO in a 1:1 ratio. The samplemixture was analyzed by AKTA Pure system with Superdex 200 HiLoad 16/60column and 180 (1 ml each) fractions were collected.

The A280 Chromatogram (FIG. 9) shows three distinct peaks; a highermolecular weight Peak A (retention volume 52.05 mL), Peak B (retentionvolume 66.87 mL) and Peak C (retention volume 75.43 mL). Each of thepeak area fractions were pooled separately; the higher molecular weightpeak area was pooled into two samples. The pooled samples: S1 (fractions46-49), S2 (fractions S1-54), S3 (fractions 65-68) and S4 (fractions74-77) were concentrated using Amicon Ultra-4 Centrifugal Filters(Ultracef 30K 30,000 NMWL ref UFC803024).

MALDI-TOF procedure: Deionized water (“diH2O”) was added (500 μL) to apre-washed Pall NanoSep filter followed by various volumes of the aboveS1, S2, S3, and S4 samples. The samples were then centrifuged at12,000×g for seven minutes and washed one more time with water andreconstituted directly with a sinapinic acid (Sigma-Aldrich 49508)trifluoroacetic acid (Alfa-Aesar 44630) matrix solution to achieve 2mg/mL protein concentration. Two microliters of each sample was directlyplotted to the MALDI-TOF plate and allowed to dry. A second spotting wasperformed to improve sensitivity. The MALDI-TOF was calibrated using aMALDI-grade BSA standard on a Shimadzu Axima Confidence MALDI-TOF withlaser power was set at 65. The MALDI-TOF result (FIG. 10) showed thatsamples S1 and S2 primarily consisted of a ˜90 KDa protein species(rTPO) and ˜150 KDa proteins representing human TPO autoantibodies.

The results showed that the interaction between the labeled rTPO and thepatient samples is specific to patient anti-TPO antibodies.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is, therefore, intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

1. A method of detecting an anti-thyroid peroxidase antibody in abiological sample from a subject, the method comprising: a) incubatingthe biological sample from the subject with: a solid support, anunlabeled recombinant cynomolgus monkey thyroid peroxidase (rTPO), and alabeled cynomolgus monkey rTPO, wherein, in the presence of theanti-thyroid peroxidase antibody, a complex comprising the labeledcynomolgus monkey rTPO, the unlabeled cynomolgus monkey rTPO, and thesolid support is formed; and b) detecting the complex, the presence ofwhich indicates the presence of the anti-thyroid peroxidase antibody inthe biological sample.
 2. The method of claim 1, wherein the labelcomprises an enzyme conjugate, a fluorescent probe, a radioactiveisotope, a chemiluminescent compound, a bioluminescent compound, or acombination thereof.
 3. The method of claim 2, wherein the labelcomprises an acridinium ester (AE) or an analog thereof.
 4. The methodof claim 1, wherein the unlabeled cynomolgus monkey rTPO is indirectlylinked to the solid support.
 5. The method of claim 4, wherein theunlabeled cynomolgus monkey rTPO is biotinylated and the solid supportcomprises streptavidin.
 6. (canceled)
 7. A method of detecting ananti-thyroid peroxidase antibody in a biological sample from a subject,the method comprising: a) incubating the biological sample from thesubject with: a solid support, a cynomolgus monkey rTPO, and ananti-human secondary antibody, wherein, in the presence of theanti-thyroid peroxidase antibody, a complex comprising the solidsupport, the cynomolgus monkey rTPO, and the anti-human secondaryantibody is formed; and b) detecting the complex, the presence of whichindicates the presence of the anti-thyroid peroxidase antibody in thebiological sample.
 8. The method of claim 7, wherein the anti-humansecondary antibody is directly or indirectly linked to the solidsupport.
 9. The method of claim 8, wherein the cynomolgus monkey rTPOcomprises a label.
 10. The method of claim 9, wherein the labelcomprises an enzyme conjugate, a fluorescent probe, a radioactiveisotope, a chemiluminescent compound, a bioluminescent compound, or acombination thereof.
 11. The method of claim 10, wherein the label is anacridinium ester (AE) or an analog thereof.
 12. The method of claim 3,wherein the acridinium ester (AE) analog is DMAE, NSP-DMAE, HQYAE, ZAE,Iso-Di-ZAE, TSP-AE, or HEG-GLU-AE.
 13. The method of claim 12, whereinthe cynomolgus monkey rTPO comprising a label is rTPO-NSP-DMAE.
 14. Themethod of claim 13, wherein the rTPO-NSP-DMAE is present at about 50ng/ml to about 2 μg/ml.
 15. The method of claim 3 or 11, wherein thecynomolgus monkey rTPO and the label are present in a buffer comprisingphosphate buffer, NaCl, EDTA, pluronic F-127, sodium azide, sorbitol,and sulfhydryl modified bovine serum albumin.
 16. The method of claim 7,wherein the cynomolgus monkey rTPO is directly or indirectly linked tothe solid support.
 17. The method of claim 16, wherein the anti-humansecondary antibody comprises a label.
 18. The method of claim 17,wherein the label comprises an enzyme conjugate, a fluorescent probe, aradioactive isotope, a chemiluminescent compound, a bioluminescentcompound, or a combination thereof.
 19. The method of claim 18, whereinthe label is an acridinium ester (AE) or an analog thereof.
 20. Themethod of claim 19, wherein the acridinium ester analog is DMAE,NSP-DMAE, HQYAE, ZAE, Iso-Di-ZAE, TSP-AE, or HEG-GLU-AE.
 21. (canceled)22. (canceled)
 23. The method of claim 7, wherein the cynomolgus monkeyrTPO is indirectly linked to the solid support.
 24. The method of claim23, wherein the cynomolgus monkey rTPO is biotinylated and the solidsupport comprises streptavidin.
 25. The method of claim 23, wherein thecynomolgus monkey rTPO and the solid support are present in a buffercomprising phosphate buffer, NaCl, EDTA, pluronic F-127, sodium azide,sorbitol, and sulfhydryl modified bovine serum albumin.
 26. (canceled)27. A method of detecting an anti-thyroid peroxidase antibody in abiological sample from a subject, the method comprising: a) incubatingthe biological sample from the subject with a solid support, anunlabeled anti-TPO antibody, a cynomolgus monkey rTPO, and a labeledanti-TPO antibody; and b) detecting the anti-thyroid peroxidase antibodyin the biological sample, the detecting comprising analyzing a decreasein the formation of a complex comprising the solid support, theunlabeled anti-TPO antibody, the cynomolgus monkey rTPO, and the labeledanti-TPO antibody.
 28. The method of claim 27, wherein the labelcomprises an enzyme conjugate, a fluorescent probe, a radioactiveisotope, a chemiluminescent compound, a bioluminescent compound, or acombination thereof.
 29. The method of claim 28, wherein the labelcomprises an acridinium ester (AE) or an analog thereof.
 30. The methodof claim 28, wherein the acridinium ester analog is DMAE, NSP-DMAE,HQYAE, ZAE, Iso-Di-ZAE, TSP-AE, or HEG-GLU-AE.
 31. The method of claim27, wherein the labeled anti-TPO antibody is anti-TPO IgG HEG-GLU-AE.32. The method of claim 31, wherein the anti-rTPO IgG HEG-GLU-AE ispresent at about 50 ng/ml to about 2 μg/ml.
 33. The method of claim 27,wherein the complex is in a buffer comprising phosphate buffer, NaCl,EDTA, pluronic F-127, sodium azide, sorbitol, and sulfhydryl modifiedbovine serum albumin.
 34. (canceled)
 35. (canceled)
 36. (canceled) 37.(canceled)
 38. (canceled)
 39. (canceled)
 40. (canceled)
 41. (canceled)42. (canceled)
 43. A method of diagnosing a thyroid disease in asubject, the method comprising: a) incubating a biological sample fromthe subject with: a solid support, an unlabeled recombinant cynomolgusmonkey thyroid peroxidase (rTPO), and a labeled cynomolgus monkey rTPO,wherein, in the presence of an anti-thyroid peroxidase antibody in thebiological sample, a complex comprising the labeled cynomolgus monkeyrTPO, the unlabeled cynomolgus monkey rTPO, and the solid support isformed; and b) diagnosing the subject with the thyroid disease if thecomplex is formed.
 44. A method of diagnosing a thyroid disease in asubject, the method comprising: a) incubating a biological sample fromthe subject with: a solid support, a cynomolgus monkey rTPO, and ananti-human secondary antibody, wherein, in the presence of ananti-thyroid peroxidase antibody in the biological sample, a complexcomprising the solid support, the cynomolgus monkey rTPO, and theanti-human secondary antibody is formed; and b) diagnosing the subjectwith the thyroid disease if the complex is formed.
 45. A method ofdiagnosing a thyroid disease in a subject, the method comprising: a)incubating a biological sample from a subject with a solid support, anunlabeled anti-TPO antibody, a cynomolgus monkey rTPO, and a labeledanti-TPO antibody; and b) diagnosing the subject with the thyroiddisease if the formation of a complex comprising the solid support, theunlabeled anti-TPO antibody, the cynomolgus monkey rTPO, and the labeledanti-TPO antibody is decreased.
 46. The method of claim 43, wherein thethyroid disease is an autoimmune disorder.
 47. The method of claim 46,wherein the autoimmune disorder is Hashimoto's thyroiditis or Graves'disease.
 48. A recombinantly produced thyroid peroxidase (rTPO)comprising the amino acid sequence of SEQ ID NO:
 1. 49. The rTPO ofclaim 48, wherein the rTPO comprises a label.
 50. The rTPO of claim 49,wherein the label comprises an enzyme conjugate, a fluorescent probe, aradioactive isotope, a chemiluminescent compound, a bioluminescentcompound, or a combination thereof.
 51. The rTPO of claim 50, whereinthe label comprises an acridinium ester (AE) or an analog thereof. 52.The rTPO of claim 51, wherein the acridinium ester analog is DMAE,NSP-DMAE, HQYAE, ZAE, Iso-Di-ZAE, TSP-AE, or HEG-GLU-AE.
 53. The rTPO ofclaim 49 in complex with an anti-thyroid peroxidase antibody.
 54. A cDNAencoding the rTPO of claim
 49. 55. A kit comprising: a solid support, anunlabeled cynomolgus monkey rTPO, and a labeled cynomolgus monkey rTPO.56. The kit of claim 55, wherein the label comprises an enzymeconjugate, a fluorescent probe, a radioactive isotope, achemiluminescent compound, a bioluminescent compound, or a combinationthereof.
 57. The kit of claim 55, wherein the solid support comprises acolumn matrix material, a culture plate, a tube, a dish, a flask, amicrotiter plate, a bead, or a combination thereof.
 58. The kit of claim55, wherein the label is an acridinium ester (AE) or an analog thereof.59. A kit comprising: a solid support; a cynomolgus monkey rTPO; and ananti-human secondary antibody.
 60. The kit of claim 59, wherein thesolid support comprises a column matrix material, a culture plate, atube, a dish, a flask, a microtiter plate, a bead, or a combinationthereof.
 61. The kit of claim 59, wherein: the cynomolgus monkey rTPOcomprises a label; or the anti-human secondary antibody comprises alabel.
 62. The kit of claim 61, wherein the label comprises an enzymeconjugate, a fluorescent probe, a radioactive isotope, achemiluminescent compound, a bioluminescent compound, or a combinationthereof.
 63. The kit of claim 62, wherein the label is an acridiniumester (AE) or an analog thereof.
 64. A kit comprising: a solid support;a cynomolgus monkey rTPO; and an anti-TPO antibody.
 65. The kit of claim64, wherein the solid support comprises a column matrix material, aculture plate, a tube, a dish, a flask, a microtiter plate, a bead, or acombination thereof.
 66. The kit of claim 64, wherein a portion of theanti-TPO antibodies are unlabeled and a portion of the anti-TPOantibodies are labeled.
 67. The kit of claim 66, wherein the labelcomprises an enzyme conjugate, a fluorescent probe, a radioactiveisotope, a chemiluminescent compound, a bioluminescent compound, or acombination thereof.
 68. The kit of claim 67, wherein the label is anacridinium ester (AE) or an analog thereof.